Epithelial barrier function and cellular respiration are key cellular phenotypes in health and disease and as such involved in the progression of many pathological disorders. Accordingly, the molecular drivers are targeted extensively in drug development using appropriate disease models in vitro. So far, quantification of barrier function and metabolic respiration had to be conducted in individual phenotypic assays, making it impossible to track changes simultaneously in a single cell layer over longer periods. We have developed an assay platform that allows for simultaneous monitoring of both, the epithelial barrier function and metabolic activity of cell layers cultured on permeable substrates label-free and non-invasively. Therefore, we designed a stainless-steel measurement chamber capable of combining impedance spectroscopy and ratiometric fluorescence-based oxygen mapping. In this platform, the barrier function is quantified as the transepithelial electrical resistance (TER) while the respiratory activity is expressed as the apparent oxygen consumption rate (AOCR) yielding the name TER-Ox for the combined setup. We validated the TER-Ox system by studying the epithelial cell lines MDCK-I, MDCK-II, and A549, covering a wide range of barrier tightness. Results of the combined TER-Ox setup were compared to established but individual readouts of barrier function (cellZscope®) and oxygen consumption (VisiSens TD®). Also, we show that differences in both parameters are readily monitored while treating cell layers with modulators affecting the electron transport chain (Antimycin A and malonoben) or barrier integrity (Cytochalasin D).